Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine

Cumming, Dale A.; Hellerqvist, Carl G.; Harris-Brandts, Marees; Michnick, Stephen W.; Carver, Jeremy P.; Bendiak, Brad

doi:10.1021/bi00441a051

Cited by 106 publications

(72 citation statements)

References 18 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The site-specific composition and heterogeneity data for the N-linked carbohydrates reported here are consistent with previous reports (Green et al, 1988;Townsend et al, 1988;Cumming et al, 1989;Rice et al, 1990). Compositions consistent with triantennary Asn-linked carbohydrates with the general structure (Ne~Ac)~(HexH~x N A c )~H~x , H~x N A c~ were identified at Ams1, and AsnI5'.…”

Section: Discussionsupporting

confidence: 91%

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

1993

View full text Add to dashboard Cite

A mass spectrometry method has been developed for selective detection of glycopeptides at the low (525) picomole level during chromatography of glycoprotein digests and for differentiation of 0-linked from N-linked oligosaccharides. The technique involves observation of diagnostic sugar oxonium-ion fragments, particularly the HexNAc+ fragment at m/z 204, from collisionally excited glycopeptides. Collision-induced fragmentation can be accomplished in either of two regions of a triple quadrupole mass spectrometer equipped with an atmospheric pressure, electrospray (ES) ionization source. If collisions before the first quadrupole are chosen, it is possible to enhance formation of carbohydrate-related fragment ions without distorting the distribution of peptide and glycopeptide signals by increasing the collisional excitation potential only during that portion of each scan in which the low mass carbohydrate-related ions are being detected. This procedure, requiring only a single quadrupole instrument, identifies putative glycopeptide-containing fractions in the chromatogram but suffers from a lack of specificity in the case of co-eluting peptides. Increased specificity is obtained by selectively detecting only those parent ions that fragment in 4 2 , the second collision region of the triple quadrupole, to produce an ion at m/z 204 (HexNAc'). Only (M + H)+ ions of glycopeptides are observed in these liquid chromatography-electrospray tandem mass spectrometry (LC-ESMS/MS) "parent-scan" spectra. N-linked carbohydrates are differentiated from 0-linked by LC-ESMS/MS analysis of the digested glycoprotein prior to and after selective removal of N-linked carbohydrates by peptide N:glycosidase E These methods, which constitute the first liquid chromatography-mass spectrometry (LC-MS)-based strategies for selective identification of glycopeptides in complex mixtures, facilitate location and preparative fractionation of glycopeptides for further structural characterization. In addition, these techniques may be used to assess the compositional heterogeneity at specific attachment sites, and to define the sequence context of the attachment site in proteins of known sequence. The strategy is demonstrated for bovine fetuin, a 42-kDa glycoprotein containing three N-linked, and at least three 0-linked carbohydrates. Over 90% of the fetuin protein sequence was also corroborated by these LC-ESMS studies.

show abstract

Section: Discussionsupporting

confidence: 91%

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

1993

View full text Add to dashboard Cite

show abstract

“…Fully glycosylated fetuin contains ϳ51 mols of sugar/mol of protein and is ϳ20% carbohydrate by mass. Fetuin's three N-linked sites contain bi- (19) and triantennary structures (20,21), and its three O-linked sites bear sialylated diand tetrasaccharides (22). We also used fetuin along with gp120 to examine the contribution of RC-100's circular structure and its internal disulfide ladder to binding.…”

Section: Resultsmentioning

confidence: 99%

Retrocyclin, an Antiretroviral θ-Defensin, Is a Lectin

Wang¹,

Cole²,

Hong³

et al. 2003

The Journal of Immunology

184

164

View full text Add to dashboard Cite

θ-Defensins are circular octadecapeptides that contain an internal tridisulfide ladder. Because retrocyclin-1, an ancestral hominid θ-defensin, can protect human cells in vitro from infection by T- and M-tropic strains of HIV-1, we used surface plasmon resonance techniques to study its binding to glycoproteins and glycolipids implicated in HIV-1 entry. Retrocyclin-1 bound with high affinity to gp120 (Kd, 35.4 nM), CD4 (Kd, 31 nM), and galactosylceramide (Kd, 24.1 nM). Neither a circular form of retrocyclin without its tridisulfide ladder nor its β-hairpin precursor with these disulfides intact bound gp120 or CD4 effectively. Retrocyclin also bound fetuin, an extensively glycosylated protein, with high affinity, but it did not bind nonglycosylated gp120 or BSA. However, retrocyclin did bind to a neoglycoprotein, BSA, with covalently attached sugar residues. Experiments with glycosidase-treated fetuin, gp120, and CD4 revealed that both O-linked and N-linked sugars were used as binding sites. In a panel of retrocyclin variants, binding to immobilized gp120 and CD4 were highly correlated to each other and to the peptide’s ability to protect human PBMCs from infection by HIV-1. Although small, cysteine-rich antimicrobial peptides with lectin-like properties exist in plants, θ-defensins are the first such molecules to be identified in vertebrates. Retrocyclin’s ability to recognize and bind carbohydrate-containing surface molecules is integrally related to its ability to protect cells from HIV-1 infection.

show abstract

“…To further investigate R84A and S128R ligand binding, bovine fetuin, a protein with thoroughly analyzed carbohydrate modification (35)(36)(37), was coated onto polystyrene dishes and the recombinant selectin proteins were tested for adherence. As a negative control, binding to a second protein, yeast invertase which is highly mannosylated but lacks typical mammalian complex carbohydrate modification, was also tested for selectin binding.…”

Section: Resultsmentioning

confidence: 99%

Single Amino Acid Residues in the E- and P-selectin Epidermal Growth Factor Domains Can Determine Carbohydrate Binding Specificity

Revelle¹,

Scott²,

Beck

1996

Journal of Biological Chemistry

View full text Add to dashboard Cite

E-selectin and P-selectin are two closely related vascular cell adhesion proteins. Each selectin has an amino-terminal C-type lectin domain that is thought to possess the carbohydrate binding site that binds the sialylated Lewis x antigen (sLe x or CD15s) (Neu5Ac␣2-3Gal␤1-4(Fuc␣1-3)GlcNAc). In addition to the sLe x carbohydrate, P-selectin binds sulfated proteoglycan, 3-sulfated galactosyl ceramide (sulfatide), and heparin. Both E-and P-selectin have an EGF-like (EGF) domain that is immediately adjacent to and COOH-terminal to the lectin domain. We report that mutagenic substitution of single amino acid residues in either the P-or E-selectin EGF domain can dramatically alter selectin binding to sLe x , heparin, or sulfatide. Substitution of E-and Pselectin EGF domain residue Ser 128 with an arginine results in E-and P-selectin proteins that have lost the requirement for ␣1-3-linked fucose and are thus able to bind to sialyllactosamine. A similar phenotype is reported for an E-selectin mutation within the lectin domain. Additionally, we have determined that conservative substitution of EGF domain residues 124 and 128 can alter E-selectin binding such that it is able to adhere to heparin or sulfatide and can reduce P-selectin adherence to these ligands. The distance between the substituted EGF domain amino acid residues and the primary carbohydrate binding site within the lectin domain and their relative positioning as determined by the threedimensional crystal structure of the E-selectin lectin and EGF domains (Graves, B. J., Crowther, R. L., Chandran, C., Rumberger, J. B., Li, S., Huang, D.-S., Presky, D. H., Familletti, P. C., Wolitzky, B. A., and Burns, D. K. (1994) Nature 367, 532-538) suggest that there is little direct contact between the two domains. However, we report mutant binding characteristics which indicate that selectin oligosaccharide binding may be modulated by both domains and that wild-type E-and P-selectin/ sLe x binding interactions may be significantly different from those previously hypothesized.The selectins (E-, P-, and L-selectin) make up a family of three vascular cell adhesion proteins that appear to modulate the migration of leukocytes from blood into extravascular tissue. Each of these proteins is composed of an amino-terminal lectin domain followed by an EGF 1 -like domain, variable numbers of short consensus repeats, a single membrane spanning region, and a cytoplasmic tail (for review, see Refs. 1 and 2). The lectin domain of each selectin is thought to possess the carbohydrate binding site and E-, P-, and L-selectin share approximately 70% sequence identity over each of their three individual lectin domains. The best known E-and P-selectin

show abstract

Structures of asparagine-linked oligosaccharides of the glycoprotein fetuin having sialic acid linked to N-acetylglucosamine

Cited by 106 publications

References 18 publications

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

Selective identification and differentiation of N‐and O‐linked oligosaccharides in glycoproteins by liquid chromatography‐mass spectrometry

Retrocyclin, an Antiretroviral θ-Defensin, Is a Lectin

Single Amino Acid Residues in the E- and P-selectin Epidermal Growth Factor Domains Can Determine Carbohydrate Binding Specificity

Contact Info

Product

Resources

About